title: Improved thermostability of a Bacillus subtilis esterase by domain exchange.
abstract: A moderately thermostable esterase from Geobacillus stearothermophilus (BsteE) and its homolog from Bacillus subtilis (BsubE) show a high structural similarity with more than 95% homology and 74% amino acid identity. Interestingly, their thermal stability differs significantly by 30 °C in their melting temperature. In order to identify the positions that are responsible for this difference, most of the flexible amino acids assumed to confer instability were found to be in the cap region. For this reason, a 30 amino acid long cap domain fragment containing ten differing positions derived from BsteE was incorporated into the homologous gene encoding for the more labile BsubE by spliced overlap-extension PCR. The melting temperature of the two wild-type esterases and the mutant was evaluated by circular dichroism spectroscopy, while the kinetic parameters and the stability were determined with a photometric assay. The cap domain mutant maintained its activity, with a catalytic efficiency more similar to BsteE, while it exhibited an increase of the melting temperature by 4 °C compared to BsubE. Additional point mutations based on the differences of the parent enzymes gave a further increase of the thermostability up to 11 °C compared to BsubE; however, a significant reduction in activity was observed.
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany.
authors.forename: Markus G
authors.initials: MG
authors.lastname: Gall
authors.forename: Alberto
authors.initials: A
authors.lastname: Nobili
authors.forename: Ioannis V
authors.initials: IV
authors.lastname: Pavlidis
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D016133:Polymerase Chain Reaction; D013696:Temperature
chemical_list: D004950:Esterases